IL-8 receptor antagonists

ABSTRACT

This invention relates to the novel use of dianilino squarates in the treatment of disease states mediated by the chemokine, Interleukin-8 (IL-8).

This application claims the benefit of Provisional Application No.60/207,911, filed May, 30, 2000.

FIELD OF THE INVENTION

This invention relates to a novel group of dianilino squarane compounds,processes for the preparation thereof, the use thereof in treating IL-8,GROα, GROβ, GROγ, NAP-2, and ENA-78 mediated diseases and pharmaceuticalcompositions for use in such therapy.

BACKGROUND OF THE INVENTION

Many different names have been applied to Interleukin-8 (IL-8), such asneutrophil attractant/activation protein-1 (NAP-1), monocyte derivedneutrophil chemotactic factor (MDNCF), neutrophil activating factor(NAF), and T-cell lymphocyte chemotactic factor. Interleukin-8 is achemoattractant for neutrophils, basophils, and a subset of T-cells. Itis produced by a majority of nucleated cells including macrophages,fibroblasts, endothelial and epithelial cells exposed to TNF, IL-1α,IL-1β, or LPS, and by neutrophils themselves when exposed to LPS orchemotactic factors such as FMLP. M. Baggiolini et al, J. Clin. Invest.84, 1045 (1989); J. Schroder et al, J. Immunol. 139, 3474 (1987) and J.Immunol. 144, 2223 (1990); Strieter, et al, Science 243, 1467 (1989) andJ. Biol. Chem. 264, 10621 (1989); Cassatella et al, J. Immunol. 148,3216 (1992).

Groα, GROβ, GROγand NAP-2 also belong to the chemokine α family. LikeIL-8 these chemokines have also been referred to by different names. Forinstance GROα, β, γ have been referred to as MGSAα, β and γ respectively(Melanoma Growth Stimulating Activity), see Richmond et al, J. CellPhysiology 129, 375 (1986) and Chang et al, J. Immunol 148, 451 (1992).All of the chemokines of the α-family which possess the ELR motifdirectly preceding the CXC motif bind to the IL-8 B receptor.

IL-8, Groα, GROβ, GROγ, NAP-2 and ENA-78 stimulate a number of functionsin vitro. They have all been shown to have chemoattractant propertiesfor neutrophils, while IL-8 and GROα have demonstrated T-lymphocytes,and basophiles chemotactic activity. In addition IL-8 can inducehistamine release from basophils from both normal and atopicindividuals. GRO-α and IL-8 can in addition, induce lysozomal enzymerelease and respiratory burst from neutrophils. IL-8 has also been shownto increase the surface expression of Mac-1 (CD11b/CD18) on neutrophilswithout de novo protein synthesis. This may contribute to increasedadhesion of the neutrophils to vascular endothelial cells. Many knowndiseases are characterized by massive neutrophil infiltration. As IL-8,Groα, GROβ, GROγand NAP-2 promote the accumulation and activation ofneutrophils, these chemokines have been implicated in a wide range ofacute and chronic inflammatory disorders including psoriasis andrheumatoid arthritis, Baggiolini et al, FEBS Lett. 307, 97 (1992);Miller et al, Crit. Rev. Immunol. 12, 17 (1992); Oppenheim et al, Annu.Rev. Immunol. 9, 617 (1991); Seitz et al., J. Clin. Invest. 87, 463(1991); Miller et al., Am. Rev. Respir. Dis. 146, 427 (1992); Donnely etal., Lancet 341, 643 (1993). In addition the ELR chemokines (thosecontaining the amino acids ELR motif just prior to the CXC motif) havealso been implicated in angiostasis, Strieter et al, Science 258, 1798(1992).

In vitro, IL-8, Groα, GROβ, GROγ, and NAP-2 induce neutrophil shapechange, chemotaxis, granule release, and respiratory burst, by bindingto and activating receptors of the seven-transmembrane, G-protein-linkedfamily, in particular by binding to IL-8 receptors, most notably theB-receptor, Thomas et al., J. Biol. Chem. 266, 14839 (1991); and Holmeset al., Science 253, 1278 (1991). The development of non-peptide smallmolecule antagonists for members of this receptor family has precedent.For a review see R. Freidinger in: Progress in Drug Research, Vol. 40,pp. 33–98, Birkhauser Verlag, Basel 1993. Hence, the IL-8 receptorrepresents a promising target for the development of novelanti-inflammatory agents.

Two high affinity human IL-8 receptors (77% homology) have beencharacterized: IL-8Rα, which binds only IL-8 with high affinity, andIL-8RB, which has high affinity for IL-8 as well as for GRO-α, GROβ,GROγand NAP-2. See Holmes et al., supra; Murphy et al., Science 253,1280 (1991); Lee et al., J. Biol. Chem. 267, 16283 (1992); LaRosa etal., J. Biol. Chem. 267, 25402 (1992); and Gayle et al., J. Biol. Chem.268, 7283 (1993).

There remains a need for treatment, in this field, for compounds whichare capable of binding to the IL-8 α or β receptor. Therefore,conditions associated with an increase in IL-8 production (which isresponsible for chemotaxis of neutrophil and T-cells subsets into theinflammatory site) would benefit by compounds which are inhibitors ofIL-8 receptor binding.

SUMMARY OF THE INVENTION

This invention provides for a method of treating a chemokine mediateddisease, wherein the chemokine is one which binds to an IL-8 α or βreceptor and which method comprises administering an effective amount ofa compound of Formula (I) or a pharmaceutically acceptable salt thereof.In particular the chemokine is IL-8.

This invention also relates to a method of inhibiting the binding ofIL-8 to its receptors in a mammal in need thereof which comprisesadministering to said, mammal an effective amount of a compound ofFormula (I).

Compounds of Formula (I) useful in the present invention are representedby the structure:

wherein:

-   R₁ is independently selected from the group consisting of hydrogen,    halogen, nitro, cyano, halosubstituted C₁₋₁₀ alkyl, C₁₋₁₀ alkyl,    C₂₋₁₀ alkenyl, C₁₋₁₀ alkoxy, halosubstituted C₁₋₁₀ alkoxy, azide,    (CR₈R₈)q S(O)_(t)R₄, hydroxy, hydroxy C₁₋₄ alkyl, aryl, aryl C₁₋₄    alkyl, aryloxy, aryl C₁₋₄ alkyloxy, heteroaryl, heteroarylalkyl,    heterocyclic, heterocyclic C₁₋₄alkyl,; heteroaryl C₁₋₄ alkyloxy,    aryl C₂₋₁₀ alkenyl, heteroaryl C₂₋₁₀ alkenyl, heterocyclic C₂₋₁₀    alkenyl, (CR₈R₈)qNR₄R₅, C₂₋₁₀ alkenyl C(O)NR₄R₅, (CR₈R₈)q C(O)NR₄R₅,    (CR₈R₈)q C(O)NR₄R₁₀, S(O)₃H, S(O)₃R₈, (CR₈R₈)q C(O)R₁₁, C₂₋₁₀    alkenyl C(O)R₁₁, C₂₋₁₀ alkenyl C(O)OR₁₁, (CR₈R₈)q C(O)OR₁₂, (CR₈R₈)q    OC(O) R₁₁, (CR₈R₈)qNR₄C(O)R₁₁, (CR₈R₈)q NHS(O)₂R₁₇, (CR₈R₈)q    S(O)₂NR₄R₅; or two R₁ moieties together y form O—(CH₂)_(s)O— or a 5    to 6 membered unsaturated ring;-   q is 0, or an integer having a value of 1 to 10;-   t is 0, or an integer having a value of 1 or 2;-   s is an integer having a value of 1 to 3;-   R₄ and R₅ are independently selected from the group consisting of    hydrogen, optionally substituted C₁₋₄ alkyl, optionally substituted    aryl, optionally substituted aryl C₁₋₄alkyl, optionally substituted    heteroaryl, optionally substituted heteroaryl C₁₋₄alkyl,    heterocyclic, and heterocyclicC₁₋₄ alkyl, or R₄ and R₅ together with    the nitrogen to which they are attached form a 5 to 7 member ring    which optionally comprises an additional heteroatom selected from    oxygen, nitrogen or sulfur;-   Y is independently selected from the group consisting of hydrogen,    halogen, nitro, cyano, halosubstituted C₁₋₁₀ alkyl, C₁₋₁₀ alkyl,    C₂₋₁₀ alkenyl, C₁₋₁₀ alkoxy, halosubstituted C₁₋₁₀ alkoxy, azide,    (CR₈R₈)q S(O)_(t)R₄, hydroxy, hydroxyC₁₋₄alkyl, aryl, aryl C₁₋₄    alkyl, aryloxy, arylC₁₋₄ alkyloxy, heteroaryl, heteroarylalkyl,    heteroaryl C₁₋₄ alkyloxy, heterocyclic, heterocyclic C₁₋₄alkyl; aryl    C₂₋₁₀ alkenyl, heteroaryl C₂₋₁₀ alkenyl, heterocyclic C₂₋₁₀ alkenyl,    (CR₈R₈)q NR₄R₅, C₂₋₁₀ alkenyl C(O)NR₄R₅, (CR₈R₈)q C(O)NR₄R₅,    (CR₈R₈)q C(O)NR₄R₁₀, S(O)₃H, S(O)₃R₈, (CR₈R₈)q C(O)R₁₁, C₂₋₁₀    alkenyl C(O)R₁₁, C₂₋₁₀ alkenyl C(O)OR₁₁, C(O)R₁₁, (CR₈R₈)q C(O)OR₁₂,    (CR₈R₈)q OC(O)R₁₁, (CR₈R₈)q NR₄C(O)R₁₁, (CR₈R₈)q NHS(O)₂Rd, and    (CR₈R₈)q S(O)₂NR₄R₅; or two Y moieties together form O—(CH₂)_(s)O—    or a 5 to 6 membered unsaturated ring;-   n is an integer having a value of 1 to 5;-   m is an integer having a value of 1 to 4;-   R₈ is hydrogen or C₁₋₄ alkyl;-   R₁₀ is C₁₋₁₀ alkyl C(O)₂R₈;-   R₁₁ is selected from the group consisting of hydrogen, C₁₋₄ alkyl,    optionally substituted aryl, optionally substituted aryl C₁₋₄alkyl,    optionally substituted heteroaryl, optionally substituted    heteroarylC₁₋₄alkyl, optionally substituted heterocyclic, and    optionally substituted heterocyclicC₁₋₄alkyl;-   R₁₂ is selected from hydrogen, C₁₋₁₀ alkyl, optionally substituted    aryl and optionally substituted arylalkyl;-   R₁₇ is selected from the group consisting of C₁₋₄alkyl, aryl,    arylalkyl, heteroaryl, heteroarylC₁₋₄alkyl, heterocyclic, and    heterocyclicC₁₋₄alkyl, wherein the aryl, heteroaryl and heterocyclic    rings are all optionally substituted.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of Formula (I) may also be used in association with theveterinary treatment of mammals, other than humans, in need ofinhibition of IL-8 or other chemokines which bind to the IL-8RA and RBreceptors. Chemokine mediated diseases for treatment, therapeutically orprophylactically, in animals include disease states such as those notedherein in the Methods of Treatment section.

The following terms, as used herein, refer to:

-   -   “halo”—all halogens, that is chloro, fluoro, bromo and iodo.    -   “C₂₋₅alkyl” or “alkyl”—both straight and branched chain moieties        of 2 to 5 carbon atoms, unless the chain length is otherwise        limited, including, but not limited to, methyl, ethyl, n-propyl,        iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl        and the like.    -   The term “alkenyl” is used herein at all occurrences to mean        straight or branched chain moieties of 2–10 carbon atoms, unless        the chain length is limited thereto, including, but not limited        to ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl,        1-butenyl, 2-butenyl and the like.    -   “aryl”—phenyl and naphthyl;    -   “heteroaryl” (on its own or in any combination, such as        “heteroaryloxy”, or “heteroaryl alkyl”)—a 5–10 membered aromatic        ring system in which one or more rings contain one or more        heteroatoms selected from the group consisting of N, O or S,        such as, but not limited, to pyrrole, pyrazole, furan,        thiophene, quinoline, isoquinoline, quinazolinyl, pyridine,        pyrimidine, oxazole, thiazole, thiadiazole, triazole, imidazole,        or benzimidazole.    -   “heterocyclic” (on its own or in any combination, such as        “heterocyclicalkyl”)—a saturated or partially unsaturated 4–10        membered ring system in which one or more rings contain one or        more heteroatoms selected from the group consisting of N, O, or        S; such as, but not limited to, pyrrolidine, piperidine,        piperazine, morpholine, tetrahydropyran, or imidazolidine.    -   The term “arylalkyl” or “heteroarylalkyl” or “heterocyclicalkyl”        is used herein to mean C₁₋₁₀ alkyl, as defined above, attached        to an aryl, heteroaryl or heterocyclic moiety, as also defined        herein, unless otherwise indicated.

Illustrative compounds of Formula (I) include:

-   3-(2-hydroxy-phenylamino)-4-(2-bromophenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-hydroxy-phenylamino)-4-(2,3-dichlorophenylamino)-cyclobut-3-ene-1,2-dione,-   3-(4-nitro-2-hydroxy-phenylamino)-4-phenylamino-cyclobut-3-ene-1,2-dione,-   3-(4-cyano-2-hydroxy-phenylamino)-4-phenylamino-cyclobut-3-ene-1,2-dione,-   3-(2-methoxy-benzylamino)-4-phenylamino-cyclobut-3-ene-1,2-dione,-   3-(4-cyano-2-hydroxy-phenylamino)-4-(2-bromophenylamino)-cyclobut-3-ene-1,2-dione,-   3-(4-cyano-2-hydroxy-phenylamino)-4-(2-chlorophenylamino)-cyclobut-3-ene-1,2-dione,-   3-(4-cyano-2-hydroxy-phenylamino)-4-(2,3-dichlorophenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2-phenylphenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2-methoxyphenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2-ethylphenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2-phenoxyphenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2-chlorophenylamino)-cyclobut-3-ene-1,2-dione,-   6-Chloro-3-(3,4-dioxo-2-phenylamino-cyclobut-1-enylamino)-2-hydroxy-benzenesulfonamide,-   3,4-bis-(4-cyano-2-hydroxyphenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2,3-dimethylphenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2-methyl-4-bromophenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2-propylphenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2-methyl-3-chlorophenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2,3-dimethoxyphenylamino)-cyclobut-3-ene-1,2-dione,-   3-(2-Hydroxy-phenylamino)-4-(2-chloro-3-methylphenylamino)-cyclobut-3-ene-1,2-dione,-   3-(4-nitro-2-hydroxy-phenylamino)-4-(2-bromophenylamino)-cyclobut-3-ene-1,2-dione,-   3-(4-nitro-2-hydroxy-phenylamino)-4-(2-chlorophenylamino)-cyclobut-3-ene-1,2-dione,-   3,4-bis-(4-nitro-2-hydroxyphenylamino)-cyclobut-3-ene-1,2-dione,-   3-[(2-Hydroxy-phenyl)-methyl-amino]-4-phenylamino-cyclobut-3-ene-1,2-dione,-   3-(3-Hydroxy-pyridin-2-ylamino)-4-phenylamino-cyclobut-3-ene-1,2-dione,    or a pharmaceutically acceptable salt thereof.

Methods of Preparation

The compounds of Formula (I) may be obtained by applying syntheticprocedures, some of which are illustrated in the Schemes below. Thesynthesis provided for in these Schemes is applicable for producingcompounds of Formula (I) having a variety of different R, R₁, and arylgroups which are reacted, employing optional substituents which aresuitably protected, to achieve compatibility with the reactions outlinedherein. Subsequent deprotection, in those cases, then affords compoundsof the nature generally disclosed. Once the guanidine nucleus has beenestablished, further compounds of these formulas may be prepared byapplying standard techniques for functional group interconversion, wellknown in the art. While the schemes are shown with compounds only ofFormula (I) this is merely for illustration purposes only.

The desired aniline 6-scheme-1 can be prepared from the commerciallyavailable benzoxazolinone 1-scheme-1. Bromide 2-scheme-1 can be preparedfrom benzoxazolinone 1-scheme-1 using standard bromination conditionssuch as bromine and sodium acetate in acetic acid. Bromide 2-scheme-1can be converted to the cyanide 3-scheme-1 using standard proceduressuch as copper (I) cyanide in refluxing DMF. The amide 3-scheme-1 can beconverted to the BOC protected compound 4-scheme-1 using standardconditions such as BOC anhydride and triethylamine with a catalyticamount of dimethylaminopyridine in methylene chloride or anothersuitable organic solvent. The oxazolinone 4-scheme-1 can be converted tothe desired aniline 6-scheme-1 by first hydrolysis to the phenol5-scheme-1 using standard conditions such as potassium carbonate inmethanol followed by removal of the BOC protecting group using standardconditions such as trifluoroacetic acid in methylene chloride or anothersuitable organic solvent to give the aniline 6-scheme-1.

Compounds of structure 5 will be obtained from the commerciallyavailable dimethylether squarate 1 as outlined in Scheme 1. Intermediate3 can be obtained by reacting the diethylether squarate 1 with thedesired aniline 2 in refluxing ethanol or other suitable organicsolvent. The desired squarane compound 5 can be obtained by reactingsquarane 3 with a second aniline 4 in refluxing ethanol or othersuitable organic solvent.

SYNTHETIC EXAMPLES

The invention will now be described by reference to the followingexamples which are merely illustrative and are not to be construed as alimitation of the scope of the present invention. All temperatures aregiven in degrees centigrade, all solvents are highest available purityand all reactions run under anhydrous conditions in an argon atmosphereunless otherwise indicated.

In the Examples, all temperatures are in degrees Centigrade (° C.). Massspectra were performed upon a VG Zab mass spectrometer using fast atombombardment, unless otherwise indicated. ¹H-NMR (hereinafter “NMR”)spectra were recorded at 250 MHz using a Bruker AM 250 or Am 400spectrometer. Multiplicities indicated are: s=singlet, d=doublet,t=triplet, q=quartet, m=multiplet and br indicates a broad signal. Sat.indicates a saturated solution, eq indicates the proportion of a molarequivalent of reagent relative to the principal reactant.

3-(2,3-dichloroanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(100 mg, 0.43 mmol) in toluene (2 mL) and DMSO (1 drop) was added2,3-dichloroaniline (0.07 mL, 0.43 mmol) and reaction was stirred at110° C. overnight. The resulting solid was collected by filtration,washed with ethyl acetate to remove toluene and dried in vacuo. LC-MS(m/z) 349 (M⁺).

3-(2-bromoanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2-bromoanilne (36.9 mg,0.21 mmol) and reaction was stirred at 110° C. overnight. Reaction waspurified on HPLC (acetonitrile:water) and product was concentrated down.Solid was dried in vacuo. LC-MS (m/z) 359 (M⁻).

3-(2-phenylanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2-aminobiphenyl (36.3 mg,0.21 mmol) and reaction was stirred at 110° C. overnight. Reaction waspurified on HPLC (acetonitrile:water) and product was concentrated down.Solid was dried in vacuo. LC-MS (m/z) 357.2 (M⁺).

3-(2-methoxyanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2-methoxyaniline (0.024mL, 0.21 mmol) and reaction was stirred at 110° C. overnight. Reactionwas purified on HPLC (acetonitrile:water) and product was concentrateddown. Solid was dried in vacuo. LC-MS (m/z) 311.4 (M⁺).

3-(2-ethylanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2-ethylaniline (0.024 mL,0.21 mmol) and reaction was stirred at 110° C. overnight. Reaction waspurified on HPLC (acetonitrile:water) and product was concentrated down.Solid was dried in vacuo. LC-MS (m/z) 309 (M⁺).

3-(2-phenoxyanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2-penoxyaniline (49 mg,0.21 mmol) and reaction was stirred at 110° C. overnight. Reaction waspurified on HPLC (acetonitrile:water) and product was concentrated down.Solid was dried in vacuo. LC-MS (m/z) 373 (M⁺).

3-(2-chloroanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2-chloroaniline (0.023 mL,0.21 mmol) and reaction was stirred at 110° C. overnight. Reaction waspurified on HPLC (acetonitrile:water) and product was concentrated down.Solid was dried in vacuo. LC-MS (m/z) 315 (M⁺).

3-(2-methylanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2-methylaniline (0.023 mL,0.21 mmol) and reaction was stirred at 110° C. overnight. Reaction waspurified on HPLC (acetonitrile:water) and product was concentrated down.Solid was dried in vacuo. LC-MS (m/z) 295 (M⁺).

3-(2,3-dimethylanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2,3-dimethylaniline (0.026mL, 0.21 mmol) and reaction was stirred at 110° C. overnight. Reactionwas purified on HPLC (acetonitrile:water) and product was concentrateddown. Solid was dried in vacuo. LC-MS (m/z) 309 (M⁺).

3-(4-bromo-2-methylanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 4-bromo-2-methylaniline(40 mg, 0.21 mmol) and reaction was stirred at 110° C. overnight.Reaction was purified on HPLC (acetonitrile:water) and product wasconcentrated down. Solid was dried in vacuo. LC-MS (m/z) 373 (M⁺).

3-(2-propylanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2-propylaniline (0.030 mL,0.21 mmol) and reaction was stirred at 110° C. overnight. Reaction waspurified on HPLC (acetonitrile:water) and product was concentrated down.Solid was dried in vacuo. LC-MS (m/z) 323 (M⁺).

3-(3-chloro-2-methylanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 3-chloro-2-methylaniline(0.026 mL, 0.21 mmol) and reaction was stirred at 110° C. overnight.Reaction was purified on HPLC (acetonitrile:water) and product wasconcentrated down. Solid was dried in vacuo. LC-MS (m/z) 329 (M⁺).

3-(2,3-methoxyanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2,3-methoxyaniline (0.031mL, 0.21 mmol) and reaction was stirred at 110° C. overnight. Reactionwas purified on HPLC (acetonitrile:water) and product was concentrateddown. Solid was dried in vacuo. LC-MS (m/z) 341 (M⁺).

3-(2-chloro-3-methylanilino)-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione

To a solution of 3-ethoxy-4-(2-hydroxyanilino)-cyclobut-3-ene-1,2-dione(50 mg, 0.21 mmol) in DMSO (1.5 mL) was added 2-chloro-3-methylaniline(0.026 mL, 0.21 mmol) and reaction was stirred at 110° C. overnight.Reaction was purified on HPLC (acetonitrile:water) and product wasconcentrated down. Solid was dried in vacuo. LC-MS (m/z) 329 (M⁺).

3-(2-Hydroxyanilino)-4-(2-bromoanilino)-3-cyclobutene-1,2-dione

To bromoaniline (74 mg, 0.43 mmol) was added 214 μL of 2Mtrimethyaluminum in toluene at room temperature. The mixture was stirreduntil the gasoues evolution ceased, then was added3-ethoxy-4-(2-hydroxyanilino)-3-cyclobutene-1,2 dione (50 mg, 0.21 mmol)in 1 mL methylenechloride. The mixture was reacted at room temperaturefor overnight. The reaction mixture was quenched with water, followed byaqueous work up. The organic layer was dried over sodium sulfate andconcentrated. After Gilson HPLC, 14 mg (12%) of purified product wasobtained. LCMS(H⁺) 359

3-(2-bromoanilino)-4-(2-Hydroxy-4-cyanoanilino)-3-cyclobutene-1,2-dione

A solution of 3,4-diethoxy-3-cyclobutene-1,2-dione (1.2 mL, 8.12 mmol)and 3-hydroxy-4-aminobenzonitrile (1.25 g, 8.11 mmol) in ethanol washeated at 85° C. for overnight. A tan precipitates forms. The solid wasfiltered, and collected. 1.12 g (53%) of3-ethoxy-4-(2-hydroxy-4-cyanoanilino)-3-cyclobutene-1,2-dione wasobtained. LC/MS(H+) 259

A mixture of3-ethoxy-4-(2-hydroxy-4-cyanoanilino)-3-cyclobutene-1,2-dione (0.7 g,2.71 mmol) and 2-bromoaniline (0.47 g, 2.73 mmol) were heated in 6 mlDMSO for overnight. The reaction mixture was cooled to room temperature,and ethylacetate was added and precipitates formed. The filtrate waspartitioned between ethylacetate and water. The organic layer was driedover sodium sulfate and concentrated. After Gilson HPLC, 34 mg (3%) ofpurified product was obtained. LC/MS (H+) 386

3-(2-Bromoanilino)-4-(2-Hydroxy-4-nitroanilino)-3-cyclobutene-1,2-dione

A solution of3-ethoxy-4-(2-Hydroxy-4-nitroanilino)-3-cyclobutene-1,2-dione (0.39 g,1.4 mmol) and 2-bromoaniline (0.36 g, 2.09 mmol) in 2 mL DMSO was heatedat 110° C. for overnight. The reaction mixture was cooled to roomtemperature, and ethylacetate was added, and a precipitate formed. Thefiltrate was partitioned between ethylacetate and water. The organiclayer was dried over sodium sulfate and concentrated. Titration fromacetone and hexanes gave 106 mg (19%) of3-(2-bromoanilino)-4-(2-hydroxy-4-nitroanilino)-3-cyclobutene-1,2-dione.LCMS(H+) 404

3-(2-chloroanilino)-4-(2-hydroxy-4-nitroanilino)-3-cyclobutene-1,2-dione

A solution of3-ethoxy-4-(2-Hydroxy-4-nitroanilino)-3-cyclobutene-1,2-dione (0.38 g,1.37 mmol) and 2-chloroaniline (0.23 mL, 2.2 mmol) in 2 mL DMSO washeated at 110° C. for overnight. The reaction mixture was cooled to roomtemperature, and ethylacetate was added, and a precipitate formed. Thefiltrate was partitioned between ethylacetate and water. The organiclayer was dried over sodium sulfate and concentrated. Recrystalized fromethylacetate and hexanes, 203 mg (41%) of3-(2-chloroanilino)-4-(2-Hydroxy-4-nitroanilino)-3-cyclobutene-1,2-dionewas obtained. LCMS(H+) 360.

3-(2,3-dichloroanilino)-4-(2-Hydroxy-4-nitroanilino)-3-cyclobutene-1,2-dione

A solution of3-ethoxy-4-(2-Hydroxy-4-nitroanilino)-3-cyclobutene-1,2-dione (0.40 g,1.44 mmol) and 2,3-dichloroaniline (0.34 g, 2.10 mmol) in 2 mL DMSO washeated at 110° C. for overnight. The reaction mixture was cooled to roomtemperature, and ethylacetate was added, a precipitate formed. Thefilter cake was collected. Titration from acetone and hexanes gave 68 mg(11%) of3-(2-chloroanilino)-4-(2-Hydroxy-4-nitroanilino)-3-cyclobutene-1,2-dione.LCMS(H+) 394.

Method of Treatment

The compounds of Formula (I), or a pharmaceutically acceptable saltthereof can be used in the manufacture of a medicine for theprophylactic or therapeutic treatment of any disease state in a human,or other mammal, which is exacerbated or caused by excessive orunregulated IL-8 cytokine production by such mammal's cell, such as butnot limited to monocytes and/or macrophages, or other chemokines whichbind to the IL-8 α or β receptor, also referred to as the type I or typeII receptor.

Accordingly, the present invention provides a method of treating achemokine mediated disease, wherein the chemokine is one which binds toan IL-8 α or β receptor and which method comprises administering aneffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof. In particular, the chemokines are IL-8, GROα,GROβ, GROγ, NAP-2 or ENA-78.

The compounds of Formula (I) are administered in an amount sufficient toinhibit cytokine function, in particular IL-8, GROα, GROβ, GROγ, NAP-2or ENA-78, such that they are biologically regulated down to normallevels of physiological function, or in some case to subnormal levels,so as to ameliorate the disease state. Abnormal levels of IL-8, GROα,GROβ, GROγ, NAP-2 or ENA-78 for instance in the context of the presentinvention, constitute: (i) levels of free IL-8 greater than or equal to1 picogram per mL; (ii) any cell associated IL-8, GROα, GROβ, GROγ,NAP-2 or ENA-78 above normal physiological levels; or (iii) the presenceof IL-8, GROα, GROβ, GROγ, NAP-2 or ENA-78 above basal levels in cellsor tissues in which IL-8, GROα, GROβ, GROγ, NAP-2 or ENA-78respectively, is produced.

The compounds of Formula (I), in generally have been shown to have alonger t_(1/2) and improved oral bioavailabilty over the compoundsdisclosed in WO 96/25157 and WO 97/29743 whose disclosures areincorporated herein by reference.

There are many disease states in which excessive or unregulated IL-8production is implicated in exacerbating and/or causing the disease.Chemokine mediated diseases include psoriasis, atopic dermatitis, osteoarthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonarydisease, adult respiratory distress syndrome, inflammatory boweldisease, Crohn's disease, ulcerative colitis, stroke, septic shock,multiple sclerosis, endotoxic shock, gram negative sepsis, toxic shocksyndrome, cardiac and renal reperfusion injury, glomerulonephritis,thrombosis, graft vs. host reaction, alzheimers disease, allograftrejections, malaria, restinosis, angiogenesis, atherosclerosis,osteoporosis, gingivitis and undesired hematopoietic stem cells releaseand diseases caused by respiratory viruses, herpesviruses, and hepatitisviruses, meningitis, herpes encephalitis, CNS vasculitis, traumaticbrain injury, CNS tumors, subarachnoid hemorrhage, post surgical trauma,interstitial pneumonitis, hypersensitivity, crystal induced arthritis,acute and chronic pancreatitis, acute alcoholic hepatitis, necrotizingenterocolitis, chronic sinusitis, uveitis, polymyositis, vasculitis,acne, gastric and duodenal ulcers, celiac disease, esophagitis,glossitis, airflow obstruction, airway hyperresponsiveness,bronchiolitis obliterans organizing pneumonia, bronchiectasis,bronchiolitis, bronchiolitis obliterans, chronic bronchitis, corpulmonae, dyspnea, emphysema, hypercapnea, hyperinflation, hypoxemia,hypoxia, surgerical lung volume reduction, pulmonary fibrosis, pulmonaryhypertension, right ventricular hypertropy, sarcoidosis, small airwaydisease, ventilation-perfusion mismatching, wheeze and lupus.

These diseases are primarily characterized by massive neutrophilinfiltration, T-cell infiltration, or neovascular growth, and areassociated with increased IL-8, GROα, GROβ, GROγ, NAP-2 or ENA-78production which is responsible for the chemotaxis of neutrophils intothe inflammatory site or the directional growth of endothelial cells. Incontrast to other inflammatory cytokines (IL-1, TNF, and IL-6), IL-8,GROα, GROβ, GROγ, NAP-2 or ENA-78 have the unique property of promotingneutrophil chemotaxis, enzyme release including but not limited toelastase release as well as superoxide production and activation. Theα-chemokines but particularly, GROα, GROβ, GROγ, NAP-2 or ENA-78,working through the IL8 type I or II receptor can promote theneovascularization of tumors by promoting the directional growth ofendothelial cells. Therefore, the inhibition of IL-8 induced chemotaxisor activation would lead to a direct reduction in the neutrophilinfiltration.

Recent evidence also implicates the role of chemokines in the treatmentof HIV infections, Littleman et al., Nature 381, pp. 661 (1996) and Koupet al., Nature 381, pp. 667 (1996).

Present evidence also indicates the use of IL-8 inhibitors in thetreatment of atherosclerosis. The first reference, Boisvert et al., J.Clin. Invest, 1998, 101:353–363 shows, through bone marrowtransplantation, that the absence of IL-8 receptors on stem cells (and,therefore, on monocytes/macrophages) leads to a reduction in thedevelopment of atherosclerotic plaques in LDL receptor deficient mice.Additional supporting references are: Apostolopoulos, et al.,Arterioscler. Thromb. Vasc. Biol. 1996, 16:1007–1012; Liu, et al.,Arterioscler. Thromb. Vasc. Biol, 1997, 17:317–323; Rus, et al.,Atherosclerosis. 1996, 127:263–271.; Wang et al., J. Biol. Chem. 1996,271:8837–8842; Yue, et al., Eur. J. Pharmacol. 1993, 240:81–84; Koch, etal., Am. J. Pathol., 1993, 142:1423–1431.; Lee, et al., Immunol. Lett.,1996, 53, 109–113.; and Terkeltaub et al., Arterioscler. Thromb., 1994,14:47–53.

The present invention also provides for a means of treating, in an acutesetting, as well as preventing, in those individuals deemed susceptibleto, CNS injuries by the chemokine receptor antagonist compounds ofFormula (I).

CNS injuries as defined herein include both open or penetrating headtrauma, such as by surgery, or a closed head trauma injury, such as byan injury to the head region. Also included within this definition isischemic stroke, particularly to the brain area.

Ischemic stroke may be defined as a focal neurologic disorder thatresults from insufficient blood supply to a particular brain area,usually as a consequence of an embolus, thrombi, or local atheromatousclosure of the blood vessel. The role of inflammatory cytokines in thisarea has been emerging and the present invention provides a mean for thepotential treatment of these injuries. Relatively little treatment, foran acute injury such as these has been available.

TNF-α is a cytokine with proinflammatory actions, including endothelialleukocyte adhesion molecule expression. Leukocytes infiltrate intoischemic brain lesions and hence compounds which inhibit or decreaselevels of TNF would be useful for treatment of ischemic brain injury.See Liu et al., Stroke, Vol. 25., No. 7, pp. 1481–88 (1994) whosedisclosure is incorporated herein by reference.

Models of closed head injuries and treatment with mixed 5-LO/CO agentsis discussed in Shohami et al., J. of Vaisc &Clinical Physiology andPharmacology, Vol. 3, No. 2, pp. 99–107 (1992) whose disclosure isincorporated herein by reference. Treatment, which reduced edemaformation, was found to improve functional outcome in those animalstreated.

The compounds of Formula (I) are administered in an amount sufficient toinhibit IL-8, binding to the IL-8 alpha or beta receptors, from bindingto these receptors, such as evidenced by a reduction in neutrophilchemotaxis and activation. The discovery that the compounds ofFormula-(I) are inhibitors of IL-8 binding is based upon the effects ofthe compounds of Formulas (I) in the in vitro receptor binding assayswhich are described herein. The compounds of Formula (I) have been shownto be inhibitors of type II IL-8 receptors.

As used herein, the term “IL-8 mediated disease or disease state” refersto any and all disease states in which IL-8, GROα, GROβ, GROγ, NAP-2 orENA-78 plays a role, either by production of IL-8, GROα, GROβ, GROγ,NAP-2 or ENA-78 themselves, or by IL-8, GROα, GROβ, GROγ, NAP-2 orENA-78 causing another monokine to be released, such as but not limitedto IL-1, IL-6 or TNF. A disease state in which, for instance, IL-1 is amajor component, and whose production or action, is exacerbated orsecreted in response to IL-8, would therefore be considered a diseasestate mediated by IL-8.

As used herein, the term “chemokine mediated disease or disease state”refers to any and all disease states in which a chemokine which binds toan IL-8 α or β receptor plays a role, such as but not limited to IL-8,GRO-α, GRO-β, GROγ, NAP2 or ENA-78. This would include a disease statein which, IL-8 plays a role, either by production of IL-8 itself, or byIL-8 causing another monokine to be released, such as but not limited toIL-1, IL-6 or TNF. A disease state in which, for instance, IL-1 is amajor component, and whose production or action, is exacerbated orsecreted in response to IL-8, would therefore be considered a diseasestated mediated by IL-8.

As used herein, the term “cytokine” refers to any secreted polypeptidethat affects the functions of cells and is a molecule, which modulatesinteractions between cells in the immune, inflammatory or hematopoieticresponse. A cytokine includes, but is not limited to, monokines andlymphokines, regardless of which cells produce them. For instance, amonokine is generally referred to as being produced and secreted by amononuclear cell, such as a macrophage and/or monocyte. Many other cellshowever also produce monokines, such as natural killer cells,fibroblasts, basophils, neutrophils, endothelial cells, brainastrocytes, bone marrow stromal cells, epideral keratinocytes andB-lymphocytes. Lymphokines are generally referred to as being producedby lymphocyte cells. Examples of cytokines include, but are not limitedto, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8),Tumor Necrosis Factor-alpha (TNF-α) and Tumor Necrosis Factor beta(TNF-β).

As used herein, the term “chemokine” refers to any secreted polypeptidethat affects the functions of cells and is a molecule which modulatesinteractions between cells in the immune, inflammatory or hematopoieticresponse, similar to the term “cytokine” above. A chemokine is primarilysecreted through cell transmembranes and causes chemotaxis andactivation of specific white blood cells and leukocytes, neutrophils,monocytes, macrophages, T-cells, B-cells, endothelial cells and smoothmuscle cells. Examples of chemokines include, but are not limited toIL-8, GRO-α, GRO-β, GRO-γ, NAP-2, ENA-78, IP-10, MIP-1α, MIP-β, PF4, andMCP 1, 2, and 3.

In order to use a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in therapy, it will normally be formulated intoa pharmaceutical composition in accordance with standard pharmaceuticalpractice. This invention, therefore, also relates to a pharmaceuticalcomposition comprising an effective, non-toxic amount of a compound ofFormula (I) and a pharmaceutically acceptable carrier or diluent.

Compounds of Formula (I), pharmaceutically acceptable salts thereof andpharmaceutical compositions incorporating such may conveniently beadministered by any of the routes conventionally used for drugadministration, for instance, orally, topically, parenterally or byinhalation. The compounds of Formula (I) may be administered inconventional dosage forms prepared by combining a compound of Formula(I) with standard pharmaceutical carriers according to conventionalprocedures. The compounds of Formula (I) may also be administered inconventional dosages in combination with a known, second therapeuticallyactive compound. These procedures may involve mixing, granulating andcompressing or dissolving the ingredients as appropriate to the desiredpreparation. It will be appreciated that the form and character of thepharmaceutically acceptable character or diluent is dictated by theamount of active ingredient with which it is to be combined, the routeof administration and other well-known variables. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

The pharmaceutical carrier employed may be, for example, either a solidor liquid. Exemplary of solid carriers are lactose, terra alba, sucrose,talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acidand the like. Exemplary of liquid carriers are syrup, peanut oil, oliveoil, water and the like. Similarly, the carrier or diluent may includetime delay material well known to the art, such as glycerylmono-stearate or glyceryl distearate alone or with a wax.

A wide variety of pharmaceutical forms can be employed. Thus, if a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg. to about 1 g. When a liquid carrier is used,the preparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable liquid such as an ampule or nonaqueousliquid suspension.

Compounds of Formula (I) may be administered topically, that is bynonsystemic administration. This includes the application of a compoundof Formula (I) externally to the epidermis or the buccal cavity and theinstillation of such a compound into the ear, eye and nose, such thatthe compound does not significantly enter the blood stream. In contrast,systemic administration refers to oral, intravenous, intraperitoneal andintramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as liniments, lotions, creams, ointmentsor pastes, and drops suitable for administration to the eye, ear ornose. The active ingredient may comprise, for topical administration,from 0.001% to 10% w/w, for instance from 1% to 2% by weight of theFormulation. It may however comprise as much as 10% w/w but preferablywill comprise less than 5% w/w, more preferably from 0.1% to 1% w/w ofthe Formulation.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy base. The base may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives or a fattyacid such as steric or oleic acid together with an alcohol such aspropylene glycol or a macrogel. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurfactant such as a sorbitan ester or a polyoxyethylene derivativethereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable containerwhich is then sealed and sterilized by autoclaving or maintaining at98–100° C. for half an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Compounds of formula (I) may be administered parenterally, that is byintravenous, intramuscular, subcutaneous intranasal, intrarectal,intravaginal or intraperitoneal administration. The subcutaneous andintramuscular forms of parenteral administration are generallypreferred. Appropriate dosage forms for such administration may beprepared by conventional techniques. Compounds of Formula (I) may alsobe administered by inhalation, that is by intranasal and oral inhalationadministration. Appropriate dosage forms for such administration, suchas an aerosol formulation or a metered dose inhaler, may be prepared byconventional techniques.

For all methods of use disclosed herein for the compounds of Formula(I), the daily oral dosage regimen will preferably be from about 0.01 toabout 80 mg/kg of total body weight. The daily parenteral dosage regimenabout 0.001 to about 80 mg/kg of total body weight. The daily topicaldosage regimen will preferably be from 0.1 mg to 150 mg, administeredone to four, preferably two or three times daily. The daily inhalationdosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kgper day. It will also be recognized by one of skill in the art that theoptimal quantity and spacing of individual dosages of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof will bedetermined by the nature and extent of the condition being treated, theform, route and site of administration, and the particular patient beingtreated, and that such optimums can be determined by conventionaltechniques. It will also be appreciated by one of skill in the art thatthe optimal course of treatment, i.e., the number of doses of a compoundof Formula (I) or a pharmaceutically acceptable salt thereof given perday for a defined number of days, can be ascertained by those skilled inthe art using conventional course of treatment determination tests.

The invention will now be described by reference to the followingbiological examples which are merely illustrative and are not to beconstrued as a limitation of the scope of the present invention.

BIOLOGICAL EXAMPLES

The IL-8, and Gro-α chemokine inhibitiory effects of compounds of thepresent invention were determined by the following in vitro assay:

Receptor Binding Assays:

[¹²⁵I] IL-8 (human recombinant) was obtained from Amersham Corp.,Arlington Heights, Ill., with specific activity 2000 Ci/mmol. Gro-α wasobtained from NEN-New England Nuclear. All other chemicals were ofanalytical grade. High levels of recombinant human IL-8 type α and βreceptors were individually expressed in Chinese hamster ovary cells asdescribed previously (Holmes, et al., Science, 1991, 253, 1278). TheChinese hamster ovary membranes were homogenized according to apreviously described protocol (Haour, et al., J Biol. Chem., 249 pp2195–2205 (1974)). Except that the homogenization buffer was changed to10 mM Tris-HCL, 1 mM MgSO4, 0.5 mM EDTA (ethylene-diaminetetraaceticacid), 1 mM PMSF (α-toluenesulphonyl fluoride), 0.5 mg/L Leupeptin, pH7.5. Membrane protein concentration was determined using Pierce Co.micro-assay kit using bovine serum albumin as a standard. All assayswere performed in a 96-well micro plate format. Each reaction mixturecontained ¹²⁵I IL-8 (0.25 nM) or ¹²⁵I Gro-α and 0.5 μg/mL of IL-8Rα or1.0 μg/mL of IL-8Rβ membranes in 20 mM Bis-Trispropane and 0.4 mM TrisHCl buffers, pH 8.0, containing 1.2 mM MgSO₄, 0.1 mM EDTA, 25 mM NaCland 0.03% CHAPS. In addition, drug or compound of interest was addedwhich had been pre-dissolved in DMSO so as to reach a finalconcentration of between 0.01 nM and 100 uM. The assay was initiated byaddition of ¹²⁵I-IL-8. After 1 hour at room temperature the plate washarvested using a Tomtec 96-well harvester onto a glass fiber filtermatblocked with 1% polyethylenimine/0.5% BSA and washed 3 times with 25 mMNaCl, 10 mM Tris HCl, 1 mM MgSO₄, 0.5 mM EDTA, 0.03% CHAPS, pH 7.4. Thefilter was then dried and counted on the Betaplate liquid scintillationcounter. The recombinant IL-8 Rα, or Type I, receptor is also referredto herein as the non-permissive receptor and the recombinant IL-8 Rβ, orType II, receptor is referred to as the permissive receptor.

All of the exemplified compounds of Formulas (I) noted herein in theSynthetic Chemistry Section, Example 1 to 15, demonstrated an IC₅₀ fromabout 45 to about <1 μg/mL in the permissive models for IL-8 receptorinhibition. Of those compounds tested, Examples 1 to 12 were also foundto be inhibitors of Gro-α binding at about the same level.

Chemotaxis Assay:

The in vitro inhibitory properties of these compounds are determined inthe neutrophil chemotaxis assay as described in Current Protocols inImmunology, vol I, Suppl 1, Unit 6.12.3., whose disclosure isincorporated herein by reference in its entirety. Neutrophils whereisolated from human blood as described in Current Protocols inImmunology Vol I, Suppl 1 Unit 7.23.1, whose disclosure is incorporatedherein by reference in its entirety. The chemoattractants IL-8, GRO-α,GRO-β, GRO-γ and NAP-2 are placed in the bottom chamber of a 48multiwell chamber (Neuro Probe, Cabin John, MD) at a concentrationbetween 0.1 and 100 nM. The two chambers are separated by a 5 umpolycarbonate filter. When compounds of this invention are tested, theyare mixed with the cells (0.001–1000 nM) just prior to the addition ofthe cells to the upper chamber. Incubation is allowed to proceed forbetween about 45 and 90 min at about 37° C. in a humidified incubatorwith 5% CO₂. At the end of the incubation period, the polycarbonatemembrane is removed and the top side washed, the membrane then stainedusing the Diff Quick staining protocol (Baxter Products, McGaw Park,Ill., USA). Cells which have chemotaxed to the chemokine are visuallycounted using a microscope. Generally, four fields are counted for eachsample, these numbers are averaged to give the average number of cellswhich had migrated. Each sample is tested in triplicate and eachcompound repeated at least four times. To certain cells (positivecontrol cells) no compound is added, these cells represent the maximumchemotactic response of the cells. In the case where a negative control(unstimulated) is desired, no chemokine is added to the bottom chamber.The difference between the positive control and the negative controlrepresents the chemotactic activity of the cells.

Elastase Release Assay:

The compounds of this invention are tested for their ability to preventElastase release from human neutrophils. Neutrophils are isolated fromhuman blood as described in Current Protocols in Immunology Vol I, Suppl1 Unit 7.23.1. PMNs 0.88×10⁶ cells suspended in Ringer's Solution (NaCl118, KCl 4.56, NaHCO3 25, KH2PO4 1.03, Glucose 11.1, HEPES 5 mM, pH 7.4)are placed in each well of a 96 well plate in a volume of 50 ul. To thisplate is added the test compound (0.001–1000 nM) in a volume of 50 ul,Cytochalasin B in a volume of 50 ul (20 ug/ml) and Ringers buffer in avolume of 50 ul. These cells are allowed to warm (37° C., 5% CO₂, 95%RH) for 5 min before IL-8, GROα, GROβ, GROγor NAP-2 at a finalconcentration of 0.01–1000 nM was added. The reaction is allowed toproceed for 45 min before the 96 well plate is centrifuged (800 xg 5min) and 100 ul of the supernatant removed. This suppernatant is addedto a second 96 well plate followed by an artificial elastase substrate(MeOSuc-Ala-Ala-Pro-Val-AMC, Nova Biochem, La Jolla, Calif.) to a finalconcentration of 6 ug/ml dissolved in phosphate buffered saline.Immediately, the plate is placed in a fluorescent 96 well plate reader(Cytofluor 2350, Millipore, Bedford, Mass.) and data collected at 3 minintervals according to the method of Nakajima et al J. Biol Chem 2544027 (1979). The amount of Elastase released from the PMNs is calculatedby measuring the rate of MeOSuc-Ala-Ala-Pro-Val-AMC degradation.

TNF-α in Traumatic Brain Injury Assay:

The present assay provides for examination of the expression of tumornecrosis factor mRNA in specfic brain regions which followexperimentally induced lateral fluid-percussion traumatic brain injury(TBI) in rats. Adult Sprague-Dawley rats (n=42) were anesthetized withsodium pentobarbital (60 mg/kg, i.p.) and subjected to lateralfluid-percussion brain injury of moderate severity (2.4 atm.) centeredover the left temporaparietal cortex (n=18), or “sham” treatment(anesthesia and surgery without injury, n=18). Animals are sacrificed bydecapitation at 1, 6 and 24 hr. post injury, brains removed, and tissuesamples of left (injured) parietal cortex (LC), corresponding area inthe contralateral right cortex (RC), cortex adjacent to injured parietalcortex (LA), corresponding adjacent area in the right cortex (RA), lefthippocampus (LH) and right hippocampus (RH) are prepared. Total RNA wasisolated and Northern blot hybridization is performed and quantitatedrelative to an TNF-α positive control RNA (macrophage=100%). A markedincrease of TNF-α mRNA expression is observed in LH (104±17% of positivecontrol, p<0.05 compared with sham), LC (105±21%, p<0.05) and LA (69±8%,p<0.01) in the traumatized hemisphere 1 hr. following injury. Anincreased TNF-α mRNA expression is also observed in LH (46±8%, p<0.05),LC (30±3%, p<0.01) and LA (32±3%, p<0.01) at 6 hr. which resolves by 24hr. following injury. In the contralateral hemisphere, expression ofTNF-α mRNA is increased in RH (46±2%, p<0.01), RC (4±3%) and RA (22±8%)at 1 hr. and in RH (28±11%), RC (7±5%) and RA (26±6%, p<0.05) at 6 hr.but not at 24 hr. following injury. In sham (surgery without injury) ornaive animals, no consistent changes in expression of TNF-α mRNA areobserved in any of the 6 brain areas in either hemisphere at any times.These results indicate that following parasagittal fluid-percussionbrain injury, the temporal expression of TNF-α mRNA is altered inspecific brain regions, including those of the non-traumatizedhemisphere. Since TNF-α is able to induce nerve growth factor (NGF) andstimulate the release of other cytokines from activated astrocytes, thispost-traumatic alteration in gene expression of TNF-α plays an importantrole in both the acute and regenerative response to CNS trauma.

CNS Injury Model for IL-β mRNA

This assay characterizes the regional expression of interleukin-1β(IL-1β) mRNA in specific brain regions following experimental lateralfluid-percussion traumatic brain injury (TBI) in rats. AdultSprague-Dawley rats (n=42) are anesthetized with sodium pentobarbital(60 mg/kg, i.p.) and subjected to lateral fluid-percussion brain injuryof moderate severity (2.4 atm.) centered over the left temporaparietalcortex (n=18), or “sham” treatment (anesthesia and surgery withoutinjury). Animals are sacrificed at 1, 6 and 24 hr. post injury, brainsremoved, and tissue samples of left (injured) parietal cortex (LC),correspondinig area in the contralateral right cortex (RC), cortexadjacent to injured parietal cortex (LA), corresponding adjacent area inthe right cortex (RA), left hippocampus (LH) and right hippocampus (RH)are prepared. Total RNA is isolated and Northern blot hybridization wasperformed and the quantity of brain tissue IL-1β mRNA is presented aspercent relative radioactivity of IL-1β positive macrophage RNA whichwas loaded on same gel. At 1 hr. following brain injury, a marked andsignificant increase in expression of IL-1β mRNA is observed in LC(20.0±0.7% of positive control, n=6, p<0.05 compared with sham animal),LH (24.5±0.9%, p<0.05) and LA (21.5±3.1%, p<0.05) in the injuredhemisphere, which remained elevated up to 6 hr. post injury in the LC(4.0±0.4%, n=6, p<0.05) and LH (5.0±1.3%, p<0.05). In sham or naiveanimals, no expression of IL-1β mRNA is observed in any of therespective brain areas. These results indicate that following TBI, thetemporal expression of IL-1β mRNA is regionally stimulated in specificbrain regions. These regional changes in cytokines, such as IL-1β play arole in the post-traumatic.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe area can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore the Examples herein are to beconstrued as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

1. The compound selected from the group consisting of:3-(2-hydroxy-phenylamino)-4-(2-bromophenylamino)-cyclobut-3-ene-1,2-dione,3-(2-hydroxy-phenylamino)-4-(2,3-dichlorophenylamino)-cyclobut-3-ene-1,2-dione,3-(4-nitro-2-hydroxy-phenylamino)-4-phenylamino-cyclobut-3-ene-1,2-dione,3-(4-cyano-2-hydroxy-phenylamino)-4-phenylamino-cyclobut-3-ene-1,2-dione,3-(2-methoxy-benzylamino)-4-phenylamino-cyclobut-3-ene-1,2-dione,3-(4-cyano-2-hydroxy-phenylamino)-4-(2-bromophenylamino)-cyclobut-3-ene-1,2-dione,3-(4-cyano-2-hydroxy-phenylamino)-4-(2-chlorophenylamino)-cyclobut-3-ene-1,2-dione,3-(4-cyano-2-hydroxy-phenylamino)-4-(2,3-dichlorophenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2-phenylphenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2-methoxyphenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2-ethylphenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2-phenoxyphenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2-chlorophenylamino)-cyclobut-3-ene-1,2-dione,6-Chloro-3-(3,4-dioxo-2-phenylamino-cyclobut-1-enylamino)-2-hydroxy-benzenesulfonamide,3,4-bis-(4-cyano-2-hydroxyphenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2,3-dimethylphenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2-methyl-4-bromophenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2-propylphenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2-methyl-3-chlorophenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2,3-dimethoxyphenylamino)-cyclobut-3-ene-1,2-dione,3-(2-Hydroxy-phenylamino)-4-(2-chloro-3-methylphenylamino)-cyclobut-3-ene-1,2-dione,3-(4-nitro-2-hydroxy-phenylamino)-4-(2-bromophenylamino)-cyclobut-3-ene-1,2-dione,3-(4-nitro-2-hydroxy-phenylamino)-4-(2-chlorophenylamino)-cyclobut-3-ene-1,2-dione,3,4-bis-(4-nitro-2-hydroxyphenylamino)-cyclobut-3-ene-1,2-dione,3-[(2-Hydroxy-phenyl)-methyl-amino]-4-phenylamino-cyclobut-3-ene-1,2-dione,3-(3-Hydroxy-pyridin-2-ylamino)-4-phenylamino-cyclobut-3-ene-1,2-dione,and or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising an effective amount of a compound according toclaim 1, and a pharmaceutically acceptable carrier or diluent.
 3. Amethod of treating a chemokine mediated disease state, wherein thechemokine binds to an IL-8 α or β receptor in a mammal, which comprisesadministering to said mammal an effective amount of a compound of theformula according to claim
 1. 4. The method according to claim 3 whereinthe mammal is afflicted with a chemokine mediated disease selected fromatopic dermatitis, osteo arthritis, rheumatoid arthritis, asthma,chronic obstructive pulmonary disease, adult respiratory distresssyndrome, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, stroke, septic shock, multiple sclerosis, endotoxic shock,psoriasis, gram negative sepsis, toxic shock syndrome, cardiac and renalreperfusion injury, glomerulonephritis, thrombosis, graft vs. hostreaction, alzheimers disease, allograft rejections, malaria, restinosis,angiogenesis, atherosclerosis, osteoporosis, gingivitis and undesiredhematopoietic stem cells release, diseases caused by respiratoryviruses, herpesviruses, and hepatitis viruses, meningitis, herpesencephalitis, CNS vasculitis, traumatic brain injury, CNS tumors,subarachnoid hemorrhage, post surgical trauma, cystic fibrosis, pre-termlabour, cough, pruritus, interstitial pneumonitis, hypersensitivity,crystal induced arthritis, lyme arthritis, fibrodysplasia ossificansprogressiva, acute and chronic pancreatitis, acute alcoholic hepatitis,necrotizing enterocolitis, chronic sinusitis, uveitis, polymyositis,vasculitis, acne, gastric and duodenal ulcers, celiac disease,esophagitis, glossitis, airflow obstruction, airway hyperresponsiveness,bronchiolitis obliterans organizing pneumonia, bronchiectasis,bronchiolitis, bronchiolitis obliterans, chronic bronchitis, corpulmonae, dyspnea, emphysema, hypercapnea, hyperinflation, hypoxemia,hypoxia, surgerical lung volume reduction, pulmonary fibrosis, pulmonaryhypertension, right ventricular hypertropy, sarcoidosis, small airwaydisease, ventilation-perfusion mismatching, wheeze and lupus.